Anticancer Activity of Rhizophora mucronata Leaves Extract on Sprague–Dawley Rats: In Vivo Model

Medicinal plants are now used to treat cancer due to the presence of bioactive compounds. Apart from the plants, mangroves also possess rich bioactive compounds with high medicinal activity. Based on the ethnobotanical attributes of Rhizophora mucronata, we are keen to work with its anticancer activity. The aim of the study is to assess the anticancer activity of methanolic extract of Rhizophora mucronata leaves against breast cancer. Its safety profile for anticancer investigations was therefore confirmed through an acute toxicity assessment. In accordance with OECD guiding principles, the study was approved to evaluate the toxicity, including acute and subacute effects and anticancer activities of methanolic extract of Rhizophora mucronata leaves on Sprague–Dawley rats. In acute toxicity trials, the dose of 1000 mg/kg body weight was determined to be safe and nontoxic even at high dose levels and did not result in any indicators of toxicity or death in the tested groups compared to controls for 14 days. In contrast, rats in a subacute toxicity study were given consistent doses of 100 mg/kg, 200 mg/kg, and 300 mg/kg for a total of 28 days along with a control group. Haematological, biochemical, and histological tests conducted in advance revealed that methanolic extract of Rhizophora mucronata leaves (MERML) at repeated doses of 200 mg/kg and 300 mg/kg was normal and had no significant effects on the treated groups. Rhizophora mucronata extract (250 mg/kg) was successfully used in in vivo trials to stop the growth of breast cancer cells in groups that had been given DMBA. Based on these findings, it has been concluded that methanolic extract of Rhizophora mucronata leaves (MERML) was safe at both higher and lower dosages and could be assessed for pharmacological study.


Introduction
Te disease of tissue growth directive failure cancer is a vast collection of illnesses.Te genes that control cell growth and division have to be distorted for a common cell to change into a tumour cell [1].Cancer is the subject of intense anxiety and is forbidden, and 200 diferent varieties of cancer concern humans, despite the fact that many diseases may have a prognosis that is much worse than most cases of cancer [2].Cancer has a wide variety of complex, poorly understood causes.Numerous factors, such as smoking, eating particular foods, contracting certain illnesses, being exposed to radiation, not engaging in sexual activity, being overweight, and environmental contaminants, are known to increase the risk of developing cancer [3].
Natural commodities from plants have been used for the treatment of diverse diseases from ancient times.Rhizophora mucronata belongs to the family Rhizophoraceae.It is commonly known as the loop root mangrove, red mangrove, and Asiatic mangrove.Te natural habitats of Rhizophora mucronata are estuaries, tidal creeks, and fat coastal areas.Te specifc medicinal properties of Rhizophora mucronata depend on the part employed and fnds its use to treat human health problems.Based on literature studies, Rhizophora mucronata was traditionally used to treat a variety of conditions, including leprosy, diabetes, hypertension, constipation, menstrual disorders, fever, haematuria, ulcers, haemorrhage, diarrhoea, nausea, and fever.Te purpose of the present study plan was to examine the anticancer effectiveness of methanolic extract of Rhizophora mucronata leaves (MERML) in Sprague-Dawley rats that had mammary carcinoma caused by DMBA.

Materials and Methods
2.1.Sample Collection.Rhizophora mucronata leaves were collected, authenticated and dried in shade, and powdered and soaked in methanol for 7 days.500 gms of powdered leaves of Rhizophora mucronata were extracted with 1500 ml of methanol solvents using the Soxhlet extraction method for 25 hrs.Ten, the methanolic extract of Rhizophora mucronata leaves (MERML) was fltered to obtain resolute volume and analysed for further studies such as various phytochemicals and detection of bioactive compounds using the GC-MS analysis.

Experiment Animals. Healthy young female adult
Sprague-Dawley rats used for acute, subacute, and toxicity studies, weighing 150-200 gm, aged between 6 and 8 weeks old weeks, were obtained from Biogen, Bangalore.Te experiments were conducted at the Animal House Facility of C.L. Baid Metha College of Pharmacy, Department of Pharmacology, Toraipakkam, Chennai-600 097, Tamil Nadu, India.Te experimental research proposals were authorized by the Institutional Animal Ethics Committee (IAEC), CPCSEA, New Delhi, and received an approval IAEC no.10/321/PO/Re/S/01/CPCSEA dated 11/10/2019.Also, the work was carried out through recent guidelines for the concern of laboratory animals.Te rats were adaptive toward laboratory ambience for 7 days prior to carry out the research.Te obtained rats were maintained in room temperature at 22 °C ± 3 °C under natural light/dark cycle, with relative moisture of slightest 30% and not more than 70%.During this process, rats were housed in cages and provided with pellet diet and clean water.

Acute Oral Toxicity Studies.
Te study [4] was performed using 12 female Sprague-Dawley rats divided into two groups and put into the cages.Group I (6 females) of SD rats were administrated with distilled water since they act as control group (Table 1).Also, to the Group II (6 females) of SD rats, the MERML extract is suspended in tween 80 and administered in an oral dose by gavage with a feeding pointer.Te quantity stage of 1000 mg/kg body mass was managed as well as the nourishment for the rats is limited for more 3-4 hours.Besides the amount, the animals were checked for the parameters such as skin modifcation, mobility, assertive behaviour, and understanding sound and pain, as well as respiratory actions.Troughout the complete 14 days of study, the animals were observed closely for behaviour signs, body mass, and food and water intake.

Subacute Oral Toxicity Studies.
Based on OECD guidelines-407, 28-day oral toxicity study was carried out in SD rats.In brief, 24 young Sprague-Dawley rats were selected with body weight of 150− 200 gm and divided into four groups (Table 2).Six female animals are used for each group.Group I acts as the vehicle control and groups II, III, and IV are for the assessment of the MERML.Te acute oral toxicity served as the basis for choosing the three dosing levels.Te low dose (100 mg/animal), medium dose (200 mg/animal), and high dose (300 mg/animal) were calculated from the acute toxicity dose (1000 mg).
Te rats were fed with standard food and water during the experiment.Te studied animals were monitored daily for any changes in clinical indications or death after extract administration until the experiment was over.Every experimental rat's body weight was noted on days 1, 7, 14, and 28 of the study.Food and water consumptions were calculated for both the control and treatment groups in the same manner.Troughout the entire study, the mortality of all the animals in the control and test groups (low, mid, and high) was monitored twice a day.Te animals were given isofurane anaesthesia on the 28th day, and a necropsy study was carried out on every animal.For the biochemical investigations, blood samples were taken from the rat's orbital sinus region using sodium heparin (200 IU/ml).Additionally, blood samples were obtained for haematological examinations in potassium EDTA (1.5 mg/ml).Blood samples were centrifuged at 3000 rpm for 10 minutes and used for haematological and biochemical investigations.Te organs such as the spleen, liver, and kidney were excised immediately and washed with ice-cold physiological saline and blotted dry.A part of the tissues from the spleen, liver, and kidney was removed and fxed in 10% formalin for histopathological study.

In Vivo Anticancer Studies.
Four sets of Sprague-Dawley rats, each with six female animals, were used to study the anticancer efects of MERML extract on mammary carcinoma-induced animals.Group I animals serve as the vehicle control and Group II animals as mammary cancer , which is mixed with 0.5 ml of olive oil with 0.5 ml of saline and administered through gastric intubation to cause breast cancer.Te rats were also given a single dosage of alpha-tocopherol (200 mg/rat) following the intragastric administration of DMBA for 25 consecutive days [5].Group IV animals act as positive control administrated with fuorouracil 20 mg/kg body weight (Table 3).Each rat's body weight was recorded before being sacrifced at the end of the experiment.Te heart puncture method was used to obtain the blood sample, which was then placed in centrifuge tubes with EDTA.After allowing the blood to coagulate, the serum was separated for biochemical and haematological analysis by centrifugation at 1000 g for 10 min at 4 °C.Te liver, kidney, and mammary tissue were immediately removed from sacrifced animals and carefully cleansed in ice-cold saline before being carefully blotted dry.For histological analysis, a tiny portion of the tissues from the liver, kidney, and mammary tissue was taken and preserved in 10% formalin.

Haematological Investigations.
Using a SYSMEX Kx-21 (Eraba, Transasia) automatic haematology analyzer, the whole blood sample was analysed for the estimate of haematological parameters such as haemoglobin (Hb), red blood cells (RBCs), white blood cells (WBCs), packed cell volume (PCV), and platelets count.

Biochemical Investigations.
Te anticoagulated blood serums were analysed for biochemical parameters such as blood metabolite estimates for urea, creatinine, uric acid, protein, and albumin, as well as lipid profling for total cholesterol and triglycerides.Additionally, measurements of enzymatic and nonenzymatic antioxidants, including the activities of catalase, glutathione peroxidase, superoxide dismutase, and lipid peroxidation (LPO), as well as liver function markers, such as aspartate transaminase and alanine transaminase, were studied.

Histopathological Investigations.
Haematoxylin and eosin staining was used for the histopathological research [6].Histopathological tests were conducted on the dissected organs, such as the breast tissue, liver, and kidney, from all four groups of rats (Groups I, II, III, and IV).For 48 hours, the tissues were kept in 10% normal saline.Following a 24hour period, the tissues were dehydrated by using ethyl alcohol-water with varying concentrations (50, 80, and 95%) before being incubated in alcohol.Also, xylene is used to cleanse the tissues from the alcohol.Also, next, using molten parafn, the tissues were imbedded in the "L" moulds.Te tissues were divided into sections of approximately 4-6 micron thickness using a rotary microtome.After cutting them into smaller pieces, the sections were stained with haematoxylin and eosin and mounted in neutral deparafnization xylene (DPX) media for microscopic examination of the cells for any signs of necrosis and lipid alterations, etc. Axiostar plus microscope was used to take photographs with a Canon 10.1 megapixel digital camera from Japan (Zeiss, Germany).

Statistical Analysis.
GraphPad Prism version 5 was used for all statistical calculations.Te outcomes of the experiment were presented as mean SD.One-way analysis of variance (ANOVA) was used to examine the data, and then the post hoc Dunnett's test was employed to determine statistical signifcance.

Results
From Pichavaram Mangrove forest, fresh Rhizophora mucronata leaves were collected and authenticated with reference number PARC/2020/4352 by Prof. P. Jayaraman at Plant Anatomy Research Centre, West Tambaram, Chennai, Tamil Nadu.Te Rhizophora mucronata leaves were cleaned, shade dried, powdered, and extracted using a methanol solvent by the Soxhlet extraction method.Te yield of resultant extract (MERML) was 2.64 gm and used for further analysis.Te extract has various phytoconstituents and the compound identifcation was recognized by using the GC-MS analysis, exposing nine compounds [7].Te stored extract was used for toxicity studies.

Acute Oral Toxicity Study.
Group I rats served as the control group and Group II rats as the MERML treated group in the investigation on acute oral toxicity.Te animals in Group II get dosages of MERML at high concentrations of 1000 mg/kg, and the animals in Group I serve as controls without extract administration.Te behaviour of the Group I and Group II animals was tracked for 14 days.Te animals in Groups I and II are closely monitored for any behavioural changes throughout the frst four hours in both the test and control groups.Te test groups given a high dose of 1000 mg/kg showed no toxicity, mortality, or behavioural abnormalities when compared to the control animals (Table 4).
Animal body weights are recorded on days 1, 7, and 14 of the study.Te experimental animal weight in the MERML test groups was studied on days 1, 7, and 14 at 177.However, throughout the course of the 14 days, there was no discernible diference (p < 0.05).Te water and food intake for the control and MERML test groups (Group I and Group II) was computed on days 1, 7, and 14, respectively.Te Group II consumed 70.4 ± 2.3 ml of water on day 1, 70.6 ± 1.1 ml of water on day 7, and 71.2 ± 2.1 ml of water on day 14.Likewise, the control groups' water consumption was 71.5 ± 1.14 on day 1, 71.32 ± 1.13 on day 7, and 72.2 ± 1.1 on day 14.(Figures 2  and 3).
Tese results imply that a higher oral dose of 1000 mg/kg does not signifcantly increase the mortality of rats.Te food and water intake is normal in both the test and control animals.Tis suggested that the MERML extract does not show toxicity in the animal groups.

Subacute Oral Toxicity Study.
From day 1 through day 28 of subacute study, the rats' body weight decreased at concentrations of 100 mg/kg, 200 mg/kg, and 300 mg/kg in comparison to the control rats.Te outcomes are displayed in Figure 4. Te statistical analysis started with a one-way ANOVA method and fnished with a Dunnett's test.However, throughout the course of the 28 days, there was no discernible diference (p > 0.05).
Tese liver function indicators, including total proteins, albumin, creatinine, triglycerides, cholesterol, alkaline phosphatase, and alanine amino transferase, were assessed.Te level of liver function parameters decreases as extract dosage is raised.Rats treated with low, mid, and high doses as well as the control group showed no discernible diferences.Figure 8 shows the outcomes.
Te serum levels of the enzyme ALT, creatinine, and total protein did not vary signifcantly, according to the biochemical assays.Te ALP value in MERML extract at 100 mg/kg demonstrated statistical signifcance up to 200 mg/kg.Despite this, Figure 9 shows the outcomes.
Te kidney, liver, and spleen histology examinations revealed no aberrant tissue, and many neutrophils and lymphocytes were also seen.Tis study states that there was no toxicity to any of the animals, for the whole 28-day period.Organ portions are shown in photographs in Figures 10-12

In Vivo Anticancer Studies.
In vivo anticancer efectiveness of MERML was studied using Sprague-Dawley rats.Group I animals serve as the control, DMBA (25 mg/kg) was used to induce breast cancer for the animals in Group II, 250 mg/kg of MERML extract was used in Group III, and 20 mg/kg of standard fuorouracil was used in Group IV.

Final Body Weight.
After receiving MERML extract, the weight of the test animals was noted.Te control group rats were found to be 321.6 ± 2.88 gm, the DMBA cancer caused rats were found to be 331.6 ± 2.88 gm, the 250 mg/kg MERML extract treated animals were found to be 338.3 ± 2.88 gm, and the standard fuorouracil treated rats were found to be 341.6 ± 2.88 gm.Te results are depicted in Figure 13.When compared to the Group II DMBA-induced animals, the body weight of the Group III animals has increased.

Journal of Toxicology
Catalase is an antioxidant enzyme, which protects the cell by splitting the two hydrogen peroxide molecules into single particle of oxygen.Te MERML extract-treated animals show 34.13 ± 1.70 and the standard control group shows 36.60 ± 1.11 values.In the control and DMBAinduced groups, the catalyse enzyme activity shows 37.03 ± 1.33 and 26.93 ± 1.30 values.Te MERML-treated Group III has a considerable increase in catalase activity when compared to the DMBA-induced Group II.
Glutathione peroxidase activity shows 5.83 ± 0.35 in the treated groups and 6.90 ± 0.26 in the standard control groups correspondingly.In the control groups, GPX activity shows 8.50 ± 0.30, and in the DMBA induced groups, GPX activity shows 3.43 ± 0.15.Te GPX activity of MERML treated groups is increased when compared to the DMBA induced groups.Tere is a small dissimilarity observed among the control groups when compared with treated groups.
Lipid per oxidation assay is used to assess the oxidative stress.In the MERML extract treated groups, the LPO values show 38.73 ± 4.35, and in standard control treated group, it shows 33.46 ± 1.36.In the control groups, the value is 28.96 ± 3.30, and in the DMBA induced groups, it is 59.43 ± 2.72.When compared to the DMBA-induced groups, the MERML treated groups' LPO activity is lower.Figure 17 displays the estimated antioxidant levels.

Histopathological Investigations.
In control groups, the histological section of the kidney shows a normal cortex with a normal medulla region.Tere is evidence of necrosis and glomeruli surrounded by intact tubules in the 25 mg/kg DMBA cancer-induced group.Tissue sections from the 250 mg/kg MERML-treated groups show normal and mild infammation.Also, there is evidence for normal and mild infammation in the 20 mg/kg fuorouracil standard control group.Te fndings demonstrate that the DMBA cancer inducer promotes necrosis in Group II rats but not in MERML extract-treated Group III rats or standard control Group IV rats. Figure 18 depicts the study of kidney sections.
In the control groups, tissue biopsies of the liver show normal hepatocytes with a normal central vein.Te aberrant lobules with focal area of fatty alteration, infammation, necrosis, congestion, and loss of certain liver parenchymal cells can be seen in the 25 mg/kg DMBA cancer caused group.Te 250 mg/kg MERML extract-treated Group III showed a normal central vein with mild infammation.Te 20 mg/kg fuorouracil standard control shows the normal central vein and sinusoids are normal.Te fndings reveal that DMBA cancer inducer promotes infammation, necrosis, and cell death in Group II rats, but not in MERML extract-treated Group III or standard control Group IV rats. Figure 19 depicts the study of liver sections.
Te normal duct lobule units with fatty tissues are visible in the mammary tissue of Group I control rats.High mitotic activity, necrosis, and reactive lymphatic follicular hyperplasia were found in the 25 mg/kg DMBA cancerinduced Group II rats.In the mammary tissue of rats treated with 250 mg/kg MERML extract, the typical arrangement of the cells in glandular pattern, which implies nuclear hyperchromasia, was discovered.Te tissue section of 20 mg/kg fuorouracil standard control rats revealed terminal duct lobule units as well as some neoplastic cells.Te fndings reveal that the DMBA cancer inducer promotes necrosis and benign tumours in Group II rats, but the MERML inhibits cancer cell growth while displaying darker nuclei in the Group III rats. Figure 20 depicts the results of mammary tissue sections.Journal of Toxicology

Discussion
In the current investigation, an acute toxicity test was conducted in animals to determine if the administration of MERML had any toxicity efects with behavioural changes, body weight, and food and drink intake.At the end of the experimental study over the following 14 days, there were no appreciable diferences between the treatment groups and the control group in the general behaviour, weight, or food intake of the rats.It is hypothesised that rats' normal growth was infuenced by the acute oral doses of MERML.Te authors of [8,9] state that changes in body weight have been indicted as a sign of adverse pharmacological and chemical efects.
According to [10], measuring food and water intake is important when examining the safety of a product intended for therapeutic purposes because an animal's physiological status and the accomplishment of the proper reaction to the medications being tested depend on an adequate intake of nutrients.Over the extent of our study, there was no discernible change in body weight, although usual amounts of food and water were consumed.
Te authors of [11,12] examined the toxicity profle of shade-dried Orthosiphon stamineus leaves and discovered that the plant did not exhibit toxicological symptoms in rodents.Te results of the current investigation proved that methanolic extract of Rhizophora mucronata leaves can be used safely.Both the control and the MERML test groups had normal values for body weight, posture, body tone, piloerection, defecation, sensitivity response, locomotion, muscular grip strength, and urination.Convulsion-related limb paralysis symptoms are not present in the MERML experimental groups and are normal in the control groups.Tere was no evidence of toxicity or rat death in the oral acute toxicity trials of MERML up to the dose of 1000 mg/kg body weight.According to [13], drugs with an oral LD 50 greater than 5000 mg/kg may be regarded as nontoxic.As a result, MERML used orally are nontoxic.
In [14], it is said that there were no signifcant changes in the weight of the body and organs after a 28-day therapy with methanolic leaves extract of T. citrina.In our study, three dose levels, low 100 mg/kg, mid 200 mg/kg, and high 300 mg/kg of MERML, were administered to animals during the subacute oral toxicity assessment.Each animal showed a typical increase in body weight with no signifcant differences between the control and treatment groups.Animals in the low dose, mid dose, and high dose treatment groups in our study did not exhibit any appreciable alterations.
According to [15], the haematopoietic system is one of the important indicators of clinical and physiological status in rats.Te change in the test-treated and control animals was visible in the RBC records, MCV, MCH, and MCHC.Te importance of determined blood indices in analysing weaknesses has been taken into consideration [16].In our Te histopathological studies of Terminalia chebula on the spleen, liver, and kidney have lately demonstrated similar actions and have been used as excellent therapeutic agents, as suggested by [17].In our study, the histological analysis of the organs further supported these impressions.Based on the fndings, we came to the conclusion that the MERML was nontoxic, safer, and suitable for pharmacological and therapeutic uses.
According to [18], Wistar rats that were 50-56 days old were administered a single dosage of 80 mg/kg DMBA intragastrically to stimulate mammary growths.Rats in this age range become extremely vulnerable to cancer-causing substances and tumour growth because of the active expansion of the terminal ducts in breast tissue.Tis result is consistent with studies [19] that found similar results with DMBA in female Sprague-Dawley rats.In our work, breast cancer in SD rats was induced by DMBA.
In our study, the ability of the MERML to reduce the high levels of ALP in DMBA-induced rats suggests that it may be able to stabilise and exchange metabolites across cell membranes and the fusion of protein and glycogen metabolism.Also, the treatment with MERML inhibits DMBAinduced rats, and the diference appears to be statistically signifcant (p < 0.05).Te high levels of ALP found in cancer-induced animals are feature to tumour and metastasis.Te actions of liver marker enzymes are related to the level of tumour growth and can be used as a sign for analysis of cancer.Te activity of liver marker enzymes is signifcantly restored in MERML treated mice when compared with tumour induced Group II animals to that of normal control Group I animals.Furthermore, A. comosus peel extract is also proved to ameliorate the efect of DMBA induced mammary carcinoma [21].
Zingue et al. [22] demonstrated that DMBA alters the normal process of mammary gland diferentiation of terminal ducts into alveoli and lobules.In our study, the DMBA-induced mammary tissue sections show the diferentiation of terminal ducts.Additionally, the histological analysis of MERML treatment animals' mammary tissue sections shows the adverse efect in inhibiting the growth of cancer cells.However, the kidney and liver tissue sections did not show any signifcant changes.
Te in vivo anticancerous properties of the methanolic leaves extract of Rhizophora mucronata (MERML) are well analysed.Notably, favonols present in the MERML exhibits the cancer-preventive properties for targeting mammary gland tissue.Te bioactive component present in the methanolic leaf extract of Rhizophora mucronata may be a viable candidate for the development of a pharmaceutically efective breast cancer treatment medication.

Conclusion
In conclusion, throughout the experimental procedures, there was no mortality of rats, and no signifcant alterations were seen in the control and MERML dose-treated groups in the acute, subacute, and anticancer investigation.Tis established that MERML does not show the toxicity to the animals and it is safe to use.Due to the presence of various phytochemicals, MERML extract shows a competent antagonistic efect against breast cancer.Based on the in vivo testing, it was recognized that the methanolic leaves extract of Rhizophora mucronata has the infuence to inhibit the growth of breast cancer.Furthermore, the isolation and purifcation of bioactive substances from Rhizophora mucronata leaves may reveal the presence of potent anticancer compound.Tus, Rhizophora mucronata leaves possess anticancer properties against the breast cancer in the feld of herbal medicine. .

Figure 7 :
Figure7: Graph shows the haematology observation of SD rats exposed to methanol extract of R. mucronata leaves.Low dose, 100 mg/kg, mid dose, 200 mg/kg, and high dose, 300 mg/kg.

Figure 17 :Figure 18 :
Figure 17: Graph shows the efect of serum enzymatic and nonenzymatic antioxidant activities of SD rats exposed to methanol extract of R. mucronata leaves.Group I, control, group II, DMBA induced (25 mg/kg), group III, extract treated (250 mg/kg), and group IV, standard fuorouracil (200 mg/kg).

Table 1 :
Te animal groups specifcation in acute studies.

Table 2 :
Te animal groups specifcation in subacute studies.
weight changes are shown in Figure1.Dunnett's test is employed as one type of ANOVA in the statistical analysis.

Table 3 :
Te animal groups specifcation in anticancer studies.

Table 4 :
Te behavioural indications of group I and group II animals.
Graph shows the water intake (gm/day) of SD rats exposed to methanol extract of R. mucronata leaves.Low dose, 100 mg/kg, mid dose, 200 mg/kg, and high dose, 300 mg/kg.
Figure 4: Graph shows the change in body weight (mg/kg) of SD rats exposed to methanol extract of R. mucronata leaves.Low dose, 100 mg/kg, mid dose, 200 mg/kg, and high dose, 300 mg/kg.Figure 6: Graph shows the food intake (gm/day) of SD rats exposed to methanol extract of R. mucronata leaves.